Blood coagulation may be viewed as a series of linked enzymatic reactions in which proteolytic activation of plasma zymogens proceeds sequentially and results in clot formation. Calcium(II) occupies a central role as a cofactor of enzymatic proteolysis and of protein complex formation in the activation of factor IX by activated factor XI, the activation of factor X by either the venom protein of Russell's viper or a complex of factor VIII, activated factor IX, and phospholipid, and the activation of prothrombin by the complex of activated factor X, factor V, and phospholipid. Recent use of trivalent ianthanide ions as substitutes for Ca(II) has facilitated the study of Ca(II)-binding proteins. Studies of the interaction of lanthanide ions with bovine factor X have demonstrated that those ions are competitive inhibitors of Ca(II)-dependent catalysis and are cofactors in metal- dependent protein-complex formation. This approach has led to the successful affinity chromatography of the venom coagulant protein using factor X-Sepharose and lanthanide ions. We propose to extend this previous study to characterize the metal-dependent protein complexes which participate in blood coagulation. The metal-binding properties of bovine factors XIa, IX, IXa, VIII, V, II, Xa, thrombin and the prothrombin activation fragments will be studied by the steady state rate dialysis method using Gd153(III), UV absorption difference spectroscopy, and fluorescence emission spectroscopy. Similar spectroscopic approaches will be used to study possible non-productive metal protein complex formation and to characterize the enzyme-protein substrate interactions in the presence of lanthanide ions. Affinity methods will provide an approach to the study of binding specificities and cofactor requirements of protein complexes involved in blood coagulation. Affinity chromatography using lanthanide ions to inhibit catalysis and facilitate enzyme-protein substrate interaction may provide a method for obtaining quantities of purified clotting proteins for structural and functional characterization and, perhaps, for clinical use.